Interaction and integration among behaviors of adult Drosophila in nature

Living in environments whose ecologies vary in periods as short as 24 h is a challenge for animals as Drosophila species that inhabit pear and apple orchards. These orchards have sunny and shady sections. The size and shape of these habitats change daily according to the position of the sun in the sky. Sunny areas are related to dryness and water loss, and shady places have lower temperatures and higher humidity. The presence of heterospecific flies may lead to competition for space and food. In sunny habitats we did not find adult Drosophila. In shady sections we found conspecific groups D. melanogaster, D. simulans, D. immigrans, D. subobscura, and the Chilean endemic D. pavani perched on grasses and herbs at 8–10 cm from fruits that had fallen on the ground. In the fruits, 99% of the adults were females and they were not grouped. The way in which daily changes in the size and shape of shady habitats together with the presence of heterospecific adults influence the selection of places to live is poorly understood in Drosophila. Our experiments show that adults of the five species prefer dark areas. The experimental results show that the odors of each species: i) influence conspecifics to select similar perch sites and decrease mobility, and ii) increase mobility in heterospecific adults and modify their perch site preferences. Attractions between conspecifics, the repulsions between species, and preferences for shaded areas matter in choosing a place to live in the five Drosophila species. These behaviors seem to have evolved as coordinated routines, contributing to the coexistence of the five Drosophila species in the apple and pear orchards examined.


Introduction
Animal species regulate their activities in response to environmental changes, with consequences for individual biological fitness [1][2][3]. For example, changes in temperature, humidity and acidity/alkalinity on grape grains (Vitis vinifera) and prickly pear fruits (Opuntia ficusindica), modulate egg-laying site selection in Drosophila melanogaster and Drosophila simulans [4]. In some species of vertebrates, environmental changes may impact their behavior, including social relationships. Thus, chemical changes caused by anxiolytic benzodiazepine dumped a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 orchards was approximately 2 km in each locality. In the pear and apple orchards, we found that fruits fallen on the ground were in different stages of fermentation. In concordance with abundance of the decaying fruits Chilean Drosophila populations reach maximum size in the autumn [22]. We formed a strain/species/orchard/locality. That is, four strains per species, achieving a study of 20 strains in total. We examined such a number of strains and species because we wanted to know if daily changes in lighting conditions in fruit orchards affected all Drosophila species equally. The five species have among themselves different degrees of relatedness [23].
Each strain was founded with 12 males and 12 females. Stocks were all reared under lights on continuously at 18.0 ± 1.0˚C and 80.00% humidity in 250 cc glass bottles on synthetic Burdick's medium [24]. Facilities to change the light/dark cycle were not available in the laboratory. The life cycle duration of D. pavani and D. immigrans is approximately twice as that of D. melanogaster, D. simulans and D. subobscura at 18.0˚C [21]. We conveniently coordinated the culture tasks to collect adults of the five species within a maximum period of a week. The adults tested in the experiments corresponded approximately to the fourth generation of breeding in the laboratory. All adults of the strains and species were vigorous, actively moving, and female fecundities were similar as suggested by the abundant offspring.

Illumination conditions in the orchards
In the orchards there were microhabitats that differed in illumination, and they were classified as sunny and shady habitats (Fig 1). We also detected that the sun's rays were projected onto the ground through the canopy of the trees creating areas that were less illuminated than the sunny places, but more illuminated than the shady sections; those sites were called sunny/ shady habitats (Fig 1). The size and shape of the three habitats daily changed according to sun position in the sky. We measured the amount of light in the three habitats between 12:00 and 14:30 (n = 40 measurements/microhabitat/orchard/locality). We used a Leitz-Westlar Microsix-L 2875 photometer and a random number table to select the sites to perform the records [25]. During that same period of time, we recorded temperatures in sunny and shady habitats. We used a Braun PTR 2000 maximum and minimum thermometer.

Fruit collections
We recorded the number of fruits fallen on the ground under the canopy of n = 40 trees/ orchard /locality (Chillán and Quillón). We carefully inspected the individual fruits searching for adult Drosophila. Subsequently, we collected at random n = 5 pears (apples) from each of n = 40 fruit trees/orchard/locality. Each collected fruit was weighed using a Sartorius CP 225D balance.

Adult Drosophila in the fruit orchards
We used our portable telephones (iPhone XR iOS14.1) at maximum magnification to take photographs of fruits fallen on the ground. Approximately 80.00% of the fruits had fissures. In these crevices we found adult Drosophila (Fig 2). Most of the imagoes were grouped on grasses and herbs near the fruits that had fallen to the ground (see Results). Due to the poor contrast of the flies against grasses and herbs, we could not obtain reliable images with our portable telephones. There were wine cellars one block away from the apple and pear orchards. In the buildings we detected groups of adult Drosophila similar to those observed in the fruit orchards, and illumination conditions were apt to take photographs (Fig 3). Identifying the species in shady habitats was extremely difficult. We used a glass aspirator to carefully collect the fly clusters (D. melanogaster, D. simulans, D. subobscura, D. immigrans and D. pavani) observed on grasses and herbs. Each fly group was deposited into a 39.5 cm 3 labeled vial filled with 2.5 cm 3 of Drosophila Burdick's medium [24]. We also carefully collected the adults found in the fissures of the fruits fallen on the ground. By using Brncic keys [26] and a Leica M7 stereoscopic microscope, we taxonomically classified the imagoes deposited in each vial. We did not determine whether the flies were virgins. In the fruit orchards, we also looked for aggressive encounters between imagoes, similar to those reported in the laboratory [27]. The observations in the fruit orchards were performed in 1 h-windows between 9:00 am and 6:00 pm.

Experiments
The experiments were designed to better understand three phenomena related to the behavior of adult Drosophila in fruit orchards that came to our attention: i) permanency in habitats with poor illumination, ii) propensity to stay near conspecifics, and iii) tendency to remain immobile (Fig 3).
The experiments were performed at 20˚C and 80% humidity between 9.30 and 12.30 am. For each treatment we recorded n = 50 individuals sex/strain/species/fruit orchard/locality. To reduce the experimental error, the records were made alternately at random. For this we use a table of random numbers [25]. For example, we recorded the behavior of a male (or female)/ Quillón strain/D. melanogaster/pear orchard/Quillón locality. Then we recorded the behavior of a female (or male)/Chillán strain/D. subobscura/apple orchard/Chillán locality, until completing n = 50 specimens/strain/species/locality. Each author collected data in the absence of the other authors. Each team member recorded at least n = 50 specimens/sex/species/locality.
For the experiments we used 9.0 x 1.5 cm (diameter x height) Petri dishes. Each fly tested was aspirated individually into the respective Petri dish through a 0.5 cm diameter hole made in the wall of Petri dish. The hole was stoppered with a cotton wool plug to prevent flies escaping from the dish. Each Petri dish was used for one individual. We made the records 10 min after we introduced a specimen in a Petri dish. This period was sufficient for the flies to acclimate to the boxes [4]. Before performing the experiments, we checked the homogeneity of illumination conditions by using a Leitz-Westlar Microsix-L 2875 photometer. In the case of D. melanogaster, D. simulans and D. subobscura, adults tested were 4-5 days old postemergence. For D. immigrans and D. pavani, adults were 12-14 days old. In these two Drosophila species sexual maturity is reached 12 days post adult emergence from pupae [21].

Preferences for dark and illuminated sites
In the fruit orchards, adults of the five Drosophila species were in shady habitats, suggesting preferences for dark sites. We tested this conjecture by wrapping half of a glass Petri dish in thick black paper as described above (Fig 4). The lids of these Petri dishes prevented the illuminated area of the boxes from being warmer than the dark zone, without an established temperature gradient at the dividing line [28,29]. Consequently, each Petri dish had an illuminated zone and a dark sector produced by the shadow the black paper cast on the Petri dish (Fig 4).
The edge of the black paper deflected the rays of light, creating an interface zone between the dark and lighted areas of the box (Fig 4). Experiments were performed in a 2 x 3 m dark room to avoid interference from other light sources. The adults found in the dark zone presumably exhibited photonegative behavior. We assumed that the flies detected in the illuminated area had photopositive behavior. We also recorded the number of imagoes in the interface zone. We also compared the results obtained by darkening the right side of each box with those obtained by darkening the left side of the boxes. In both cases individuals tested were always found in the dark side of the boxes. We tested males and females of the strains formed with adults collected in the pear orchard (Chillán locality).

Perch and moving site preferences in Petri dishes with conspecific and strange odors
In the fruit orchards the adults were perched nearby conspecifics away from other Drosophila species (Fig 3). Conspecific odors and odors of other Drosophila species might be involved in the behavior [4,12,[30][31][32]. We used the Petri dishes described above to test whether conspecific odors and odors of another Drosophila species influenced perching site preferences and mobility. The capsules offered to the adults three sections to perch and move: ceiling, wall and floor.
In one treatment, 4 nonvirgin males and 4 nonvirgin females D. melanogaster, D. simulans, D. immigrans, D. subobscura and D. pavani were deposited, each species, into their respective Petri dish for 30 min. Four nonvirgin adults of each sex composed the clusters observed in grape orchards [4]. After 30 min, we removed the flies from the Petri dish, and one male (or female) of the same strain and species was aspirated into the corresponding Petri dish. Ten minutes later, once the specimen had acclimated to plate [4], we recorded the section (ceiling, wall, floor) where the fly tested was perched or moving.
In a second treatment, 4 nonvirgin females and 4 nonvirgin males D. subobscura remained within a Petri dish for 30 min. After this time, the adults were removed from the Petri dish, and one male (female) D. melanogaster, D. simulans, D. immigrans or D. pavani was deposited into the respective Petri dish with D. subobscura odors. Ten minutes later, we recorded the area (ceiling, wall, floor) where the fly tested was perched or moving. We used D. subobscura because after its arrival in Chile in 1980, the abundance of Chilean species of Drosophila decreased [33,34], suggesting that D. subobscura affected local Drosophila species. Preferences for sites to perch and moving of adult D. subobscura were tested in Petri dishes with their own odors and in Petri dishes with D. pavani odors.
In the two treatments described, after recording the section (ceiling, wall, floor) where each fly tested was perched or moving, we continued observing each imago for the next 2 min. We wanted to know whether the fly tested jumped from the area of the Petri dish (ceiling, wall, floor) where it had been found toward another section in the Petri dish.
The strains of D.melanogaster and D. simulans established with adults collected in apple orchards were not tested. By regulation, Jeannette Silva-López, undergraduate student at the Universidad del Bío-Bío, had a limited amount of time (10-12 months) to perform: i) openfield work, ii) experiments and iii) write a report on the results to obtain her Title of Teacher of Biology and Natural Sciences. These time constraints prevented testing all the strains.

Patterns of movement
Adults aspirated into Petri dishes appeared more active in the presence of heterospecific than conspecific odors. We recorded movement patterns (locomotion and changes in direction) of adult D. melanogaster, D. simulans, D. immigrans and D. pavani in Petri dishes with conspecific odors and in Petri dishes with D. subobscura odors. Adults of this last species were tested against their own and D. pavani odors. We recorded the individual movement pattern for 2 minutes. We used a lucid Wild M5 camera. We made the records 10 min after an adult was introduced into the Petri dish. Some flies jumped from one sector (ceiling, wall, floor) to another before the end of the 2-min observation period. We discarded these flies and replaced them with new individuals of the same strain and species. In each drawing we recorded (i) the length of the trail using a Hoffritz Map measurer; we converted the distance obtained for each track into centimeters, and (ii) the size of the turning angles using a compass.

Statistical analysis
Preferences for dark and illuminated sites. The data for preferences for dark, interface and light sites (Fig 4) were subjected to an analysis of variance. We tested adults of the Chillán and Quillón strains of D. melanogaster, D. simulans, D. immigrans, D. subobscura, and D. pavani. We applied a nested Model II ANOVA [35,36]. We also used the variances of preferences for light, dark, and interface zones and the sex x strain interaction to estimate the reliability of our records [36,37]. For this we calculate a correlation coefficient (details in [36,37]). A coefficient greater than 0.80 indicates that > 80% of the specimens tested chose between the dark, light, and interface areas of the boxes (Fig 4 and references [36,37]). We also estimated an index of concordance because J.S-L, an undergraduate student, did not have experience recording nominal behavioral events. Thus, we compared the number of agreements and disagreements between J. S-L and R. G-H regarding the assignment of the adults' preferences for the light zone, the interface and the dark zone of the Petri dishes (Fig 4). We calculated a concordance index. This index was the proportion of all occurrences in which J.S-L and R.G-H coincided. We also organized the data obtained with each strain and species in a 2 x 3 (sex x sector of Petri dish) contingency table. We tested the hypothesis that the number of individuals in the dark zone, in the light zone and in the interface was independent of sex [36].
Perch and moving site selection. For D. melanogaster and D. simulans, we tested only one strain (see Fig 5). Data obtained for each of these species were analyzed by applying a twofactor analysis of variance [36]. We evaluated the effects on perching and moving site preferences in Petri dishes with conspecific odors and D. subobscura. We also evaluated the sex of individuals tested, and the interaction between sex and odor treatment.
For data obtained with adult D. immigrans, D. pavani and D. subobscura we performed a multiway factorial analysis of variance because we recorded two strains per species. We evaluated the effects of i) conspecific and D. subosbscura odors (in the case of adult D. subosbscura we evaluated the effects of conspecific and D. pavani odors), ii) the sex of the individual tested, iii) the strain, iv) the interactions between two of these variables, and v) the sex x strain x odor treatment interaction.
Patterns of movement. A multiway ANOVA was applied to the data to test the significance of adult odors, species, strain and sex on i) length of the trail (estimator of locomotor activity) and ii) size of turn angles. The two behaviors are the principal components of adult Drosophila mobility [38,39]. We applied a correlation and regression analysis to compare the relationship between the length of the trail and the size of the angles made by the sexes in the presence of conspecific and heterospecific odors [36]. The length of the trail was chosen a priori as the independent variable because we thought that the adults showing a high rate of locomotion could also perform wider turning angles.
We used STATGRAPHICS Program, Centurion XVII 64-bit, 2014 version to make our statistical analysis.

Fruit orchards
The apple and pear orchards consisted, each orchard, of 640 trees spread over 2 acres in each orchard (Fig 1). Between the trunks of adjacent apple (pear) trees there was a distance of 10.5 ± 0.50 m. At noon the canopy of each apple (pear) tree cast a shadow of 2.25 ± 0.33 m 2 on the ground (n = 20 measurements/apple (pear) canopy/orchard/locality). The fruit orchards also had sunny areas (Fig 1). At noon the mean size of the sunny areas was 7.10 ± 0.18 m 2 (n = 20 measurements/apple (pear) orchard/locality). The sun's rays also passed through the canopies projecting onto the ground; the average size of these sunny/shady areas was 0.94 ± 0.12 m 2 at noon (Fig 1). The size and shape of the shady, sunny, and sunny/shadow areas changed daily depending on the position of the sun in the sky.
At midday, the illumination in shady areas was approximately 420 ± 13 lux (n = 20 measurements/apple (pear) orchard/locality). In sunny places illumination was estimated to be approximately 50,000 ± 88 lux, whereas in the sunny/shady sites illumination was approximately 10,000 ± 19 lux (Fig 1).

Temperatures in the orchards
The average environmental temperatures in sunny and shady habitats are in S1 Table (Chillán and Quillón localities). Measurements were made at midday. In shady places temperatures fluctuated around 24˚C, whereas in sunny habitats reach over 40˚C (S1 Table). Variances in shady sites are smaller than those calculated for sunny habitats (S1 Table).

Fruits on the ground
In the pear and apple orchards the number of fruits on the ground per fruit tree fluctuated between 5 and 8. Thus, in each orchard the potential number of rearing sites for the five Drosophila species was 5 (number of fruit units) x 640 (number of fruit trees) = 3200 breeding sites. The average weight of a Fuji apple fallen on the ground was 175.00 ± 4.30 gr (n = 40 fruit units weighed). Therefore, under each apple tree there were an average of 175 x 5 = 875 gr of fruit (or approximately 0.88 kg of Fuji apple) available for the five Drosophila species. In the case of winter Nely pears, the mean weight per fruit unit was 216 ± 3.21 gr (n = 40 fruits weighed). That is under each pear tree there were on average 1.08 kg of pear available to the five Drosophila species.

Distribution of adult Drosophila in the orchards
Between 9:00 am and 6:00 pm, adult Drosophila were in shady habitats. We did not find flies neither in sunny areas or in sunny/shady sections in the apple and pear orchards, Chillán and Quillón localities. In the shady areas, 75.42% of the adults of the five Drosophila species were on grasses and herbs at 8-10 cm from pear and apple fruits fallen on the ground. The sex ratio per species in grasses and herbs was close to 1.0 (S2 Table).
In apple and pear fruits fallen on the ground, also in shady places, we found mainly D. simulans and D. subobscura (S3 Table). Occasionally we detected D. melanogaster, D. immigrans and D. pavani. A total of 99.10% of adult Drosophila found in the fruits were females and 0.90% were males (S3 Table). The fruits showed few signs of fermentation (Fig 2), but they had fissures, presumably produced when they hit the ground. The adults were in the fissures (Fig 2).

Ethological observations of adult Drosophila perched on grasses and herbs
On grasses and herbs in the shady areas of the fruit orchards there were groups of conspecifics of the five Drosophila species (Fig 3). Adults remained immobile and rarely left the cluster, suggesting that fly exchange between groups was sporadic or absent. The flies were attentive to environmental changes. They flew in all directions because of the disturbances caused by our arrival. We discovered the fly clusters because the dark-yellowish brown, pale yellow-black, and light reddish-black colors of the thorax and abdomen of the flies contrasted with the green color of the grasses and herbs on which they were perched. The groups were 8-10 cm from apple (pear) fruits fallen on the ground. We used a glass aspirator to carefully collect the groups of flies (D. melanogaster, D. simulans, D. subobscura, D. immigrans and D. pavani). Taxonomic classification confirmed that each cluster was composed of individuals of the same species (S2 Table). Although daily changes in the sun's position modified the size and shape of shady sites, the fly groups were always detected in these habitats.

Ethological observations on fruits fallen on the ground
Apples and pears lying on the ground had fissures with a depth of 1.5 to 3.0 mm and a length of 4.1 to 5.2 cm (Fig 2). The number of fissures per fruit (apple and pear) was 1.3 ± 0.2 on average. In the fissures mainly females of D. simulans and D. subobscura were found (S3 Table). The distance between females was approximately 1.0-1.9 cm (conspecific or heterospecific females). The females remained approximately 12.0 ± 1.4 min in the fissures. On the skin of the fruits, we found 1-2 adults on average distributed here and there (Fig 2). These individuals remained immobile on the pears for approximately 5.2 ± 0.7 min and then flew in different directions. Unlike adults perched on grasses and herbs, females in the fissures could be easily collected. The fruits did not show signs of bird pecking or rodent bites (Fig 2). We did not detect aggressive encounters between flies perched on fruits (peel and fissures), grasses and herbs.

Experimental results
Response to illumination conditions. The variance ratios (F) resulting from individual preferences of males and females for the dark, interface and illuminated sections in the Petri dishes (Fig 4) D. melanogaster, D. simulans, D. immigrans, D. subobscura and D. pavani in the dark, illuminated and interface zones of Petri dishes (Fig 4). Two strains per species were tested. The strains were formed with individuals collected from shady habitats in pear orchards in Chillán and Quillón (details in Materials and Methods).

Species and strain
Petri dish zone  (Fig 4), and that < 20% represent errors of measurement, which is satisfactory for this kind of experiment [36,37]. The concordance index, the proportion of all occurrences about which J. S-L and R. G-H agreed, was 96.37%. Therefore, our method to measure the response of adult Drosophila to illumination conditions was reliable. Table 1 shows that 373 out of 500 (74.60%) males and 436 out of 500 (87.20%) females of the Chillán and Quillón strains of D. melanogaster, D. simulans, D. immigrans, D. subobscura, and D. pavani were in the dark sector of Petri dishes, whereas 27 out of 500 (5.40%) males and 25 out of 500 (5.00%) females of the same strains and species were in the illuminated zone of Petri dishes. On the other hand, 100 out of 500 (20.00%) males and 39 out of 500 (7.80%) females of the species and strains were in the interface zone ( Table 1). The chi-square goodness of fit values for the number of males in the dark, illuminated and interface zones for species and strains indicated in Table 1 were all greater than the critical value χ 2 0.05(2) = 5.99. Similar results were obtained with the females (Table 1) and the chi-square goodness of fit values were also greater than the critical value χ 2 0.05(2) = 5.99. Taken as a whole these results point to a general preference for shade places. We also tested the hypothesis that the number of individuals in the dark, illuminated and interface areas of the Petri dishes was independent of sex. In the two strains of D. simulans, numbers of females and males in the dark, illuminated and interface zones of the Petri dishes were significantly different: χ 2 = 11.48, the Chillán strain and χ 2 = 21.61, the Quillón strain, df = 2, P < 0.005. An examination of the data showed that the number of females in the dark zone exceeded the expected value (Table 1).
In D. melanogaster, D. subobscura and D. immigrans, only one population exceeded the expected frequency of females in the dark zone of the Petri dishes (Table 1) Fig 5A and 5B shows that in the Petri dishes with conspecific odors the sexes of D. melanogaster prefer the wall to perch/move (white columns). In petri dishes with odors of D. subobscura, female D. melanogaster prefer perch/move on the floor and ceiling (Fig 5A, black  columns), whereas the males perch/move on the wall, floor, and ceiling (Fig 5B, black columns). The differences between the sexes in response to D. subobscura odors were significant (F 0.05(1,93) = 4.57 for the interaction of sex and odor treatment; P < 0.05). Fig 5C and 5D shows that the sexes D. simulans prefer to perch/move on the ceiling and wall in Petri dishes with conspecific odors (white columns). In Petri dishes with D. subobscura odors (black columns), the sexes D. simulans prefer perch/move on the floor (F 0.05(1,93) = 4.33 for odor treatment; P < 0.05). The interaction of sex and odor treatment for the selection of site to perch/move was not statistically significant (F 0.05(1,93) = 2. 31; P > 0.05). Fig 5E-5H shows that approximately 60% of adults of the Chillán and Quillón strains of D. immigrans choose perch/move on the wall of Petri dishes with conspecific odors (white columns). In Petri dishes with D. subobscura odors, the sexes of the Chillán strain prefer perch/ move on the wall and floor (Fig 5E and 5F; black columns). In contrast, Quillón females of D. immigrans choose perch/move on the ceiling, wall, and floor in Petri dishes with D. subobscura odors (Fig 5G; black columns), whereas the males choose perch/move on the wall and floor (Fig 5H; black columns). The sex x strain x odor treatment interaction on perch/move site preferences of adult D. immigrans was statistically significant (F 0.05(1,192) Adults of the Chillán and Quillón strains of D. pavani choose the wall and floor to perch/ move in Petri dishes with conspecific odors (Fig 5I-5L; white columns). In the presence of D. subobscura odors (black columns), females of the two D. pavani strains prefer perch/move on the ceiling (Fig 5I and 5K), whereas the males perch/move on the ceiling and floor (Fig 5J and  5L). The interaction sex x strain x odor treatment on the perch/move sites preferences of adult D. pavani was statistically important (F 0.05(1,192) = 4.62; P < 0.05).
In Petri dishes with conspecific odors, adults of the Chillán and Quillón strains of D. subobscura choose perch/move on the wall (Fig 5M-5P; white columns). Stimulated by D. pavani odors, females of the two strains choose the wall and floor to stay/move (Fig 5M and 5O), and the males prefer the ceiling and wall (Fig 5N and 5P). The interaction sex x strain x odor treatment on the perch site preferences of adult D. subobscura was statistically significant (F 0.05 (1,192) = 4.07; P < 0.05).
After recording the sector (ceiling, wall, floor) in a Petri dish on which a fly was, we observed the specimen for another 2 min. We recorded if the fly remained in the same sector or jumped to another place in the Petri dish. In the Petri dishes with conspecific odors, not more than 6.0% of the sexes jumped toward other sections (Table 2). In Petri dishes with odors of another Drosophila species at least 12.0% of the flies jumped to other sections ( Table 2). These differences are statistically important (the G test for independence showed that the calculated Gs were all higher than the critical value χ 2 0.05(1) = 3.84). Patterns of movement. The mobility of adult Drosophila entails mainly locomotion and turns [38,39]. Table 3 Locomotion and turning angle sizes of adult D. melanogaster, D. simulans, D immigrans, D. subobscura and D pavani in Petri dishes with conspecific and strange odors a . n = 50 individuals per sex/strain/species were scored. The strains tested were formed with adults collected in pear orchards in Chillán and Quillón localities (see Materials and Methods). turning angles in Petri dishes with D. subobscura odors and decrease these behaviors in Petri dishes with conspecific odors ( Table 3). Adults of the Chillán and Quillón strains of D. subobscura also decreased locomotion and size of turns in Petri dishes with conspecific odors and increased the behaviors in Petri dishes with D. pavani odors ( Table 3). The interaction of sex x strain x species x odor treatment on locomotion and turning amplitude was statistically significant (F 0.05(4, 35) = 7.42; P < 0. 0005). Table 4 shows Pearson correlation coefficients and slopes of linear regression for angle size on adult locomotion in the Chillán and Quillón strains of D. melanogaster, D. simulans,  values were all greater than the critical value F 0.05(50,45) = 2.44. The significance of differences between slopes of linear regression within strains in the presence of conspecific odors versus strange odors yielded t (98) values greater than t (98) > 3.175 critical value, P < 0.001. The analysis indicates that the relationship between locomotion and angle size is maintained in the presence of conspecific and heterospecific odors, but slopes of the linear regression are greater in the presence of heterospecific odors. Our findings are in general agreements with those reported by York et.al. [39].

Discussion
We focused on aspects of nonsexual behavior of adults of five Drosophila species in pear and apple orchards. These fruit orchards had contrasting habitats that differed in illumination conditions: i) sunny, ii) shady, and iii) sunny/shady sites (Fig 1). Adults of D. melanogaster, D. simulans, D. immigrans, D. subobscura, and the endemic D. pavani occupied shady sections. These species formed conspecific aggregations on the ground (Fig 3). On the fruits there were no conspecific groups and most of the adults were females (S3 Table). They were in the fissures exhibited by the fruits (Fig 2); in the same fissure there could be females of two Drosophila species (S3 Table). Thus, the social interactions between the sexes of a species and between Drosophila species depend on the microenvironment they occupy. Finding mainly females in the fruits suggests an evolutionary change between the sexes of the five Drosophila species relative to the occupation of food sources.
Our experimental results agree with our observations on the adult Drosophila behavior in the fruit orchards. That is, the adults preferred dark sites to rest and move (Fig 4 and Table 1). We conjecture that i) changing lighting conditions in orchards modifying the size and shape of shady sites and ii) adult preferences for dimly lit sites influence daily movements and relocations of imagoes of the five species toward new shady sections. Higher temperatures in sunny habitats could also encourage adults to move toward shady areas [10]. Dimly lit sectors have more stable temperatures (S1 Table) and presumably higher humidity than sunny areas. Temperature and humidity influence Drosophila physiology [40]. Therefore, the shady sites could provide stability to the development of the five Drosophila species.
Our data also suggest that odors emanating from adults of the five species matter in space occupation in the fruit orchards. Thus, odors of one species i) decrease mobility and ii) favor the conspecifics selecting similar sites to perch/move (Tables 2-4, and Fig 5A-5P, white columns). Those same odors in another species i) increase adult mobility (Tables 2-4) and ii) influence each sex to choose a different perching site (Fig 5A-5P, black columns). Odors emitted by the adults of a Drosophila species decreasing mobility in their congeners and stimulating similar preferences for perching sites could be the first steps in group formation. That these same odors increase mobility and change perch site preferences in each one of the sexes of other Drosophila species could hinder the formation of mixed groups composed of two or more species. These conjectures are in alignment with it being difficult to find interspecific hybrids of Drosophila in nature [41]. However, Drosophila yakuba females and Drosophila teissieri males originate F1 progeny in nature. These hybrids exhibit, however, great reproductive and physiological deficiencies incompatible with life [42].
Why were most of the adults of the five Drosophila species in grasses and herbs and absent in fruits? Why were most of the individuals found in the fruit females? (S3 Table). Assuming that the females detected in apples and pears were nonvirgin, they could be laying their eggs. We conjectured that the odors given off by decaying fruits could attract nonvirgin females repelling males and virgin females. Fruit odors modify Drosophila social behavior [43,44]. Laying eggs in the fissures of apples and pears might help the larvae hatched from the eggs gain access to food. The absence of males and virgin males and females could be logical; they could interfere with egg laying activity. Future work should test these conjectures.
The five Drosophila species belong to different clades [23]. However, adults exhibit a similar ability and willingness to integrate responses to illumination conditions with reactions to odors from conspecifics and strangers. These findings suggest the conservation of genetic activity and neurological coordination for the integration of routines in all five species.  Table. Abundance of males and females D. melanogaster, D. simulans, D. immigrans, D. subobscura and D. pavani in apple and pear orchards located in Chillán and Quillón. The orchards had sunny, shady and sunny/shady places. Individuals listed below were perched on grasses and herbs in shady habitats in the Chillán and Quillón orchards. In sunny and sunny/ shady areas adult Drosophila was not detected. The total number of individuals per species/ orchard/locality is shown. In parentheses is the number of groups of flies per species/fruit orchard/locality (see Results). (PDF) S3 Table. Number of females and males D. simulans and D. subobscura found in fissures exhibited by apple and pear fruits fallen on the ground. The fruits were in shady environments (see Materials and Methods). The adults were collected between 9.0 am and 6.0 pm. (PDF)

Data accessibility
All data compiled by the Authors are in the Tables, Supplementary Tables and Figures that  accompany  Previous studies performed by an undergraduate student of RG-H, Jeannette Silva-López inspirated the activities in the nature and laboratory summarized in the present manuscript. The written work of Jeannette Silva-López is deposited in the Library of the Faculty of Sciences, Campus Fernando May, Universidad del Bío-Bío, City of Chillán, Chile (The Registry of Open Access Repositories). Please, write to Ms. Mónica Erazo, Head of Library, at mera-zo@ubiobio.cl for further details.